One of the goals of plant genetic engineering is to produce plants with agronomically important characteristics or traits. Recent advances in genetic engineering have provided the requisite tools to produce transgenic plants that contain and express foreign genes (Kahl et al., World J. of Microbiol. Biotech. 11:449-4160, 1995). Particularly desirable traits or qualities of interest for plant genetic engineering would include but are not limited to resistance to insects, fungal diseases, and other pests and disease-causing agents, tolerances to herbicides, enhanced stability or shelf-life, yield, environmental tolerances, and nutritional enhancements. The technological advances in plant transformation and regeneration have enabled researchers to take exogenous DNA, such as a gene or genes from a heterologous or a native source, and incorporate the exogenous DNA into the plant's genome. In one approach, expression of a novel gene that is not normally expressed in a particular plant or plant tissue may confer a desired phenotypic effect. In another approach, transcription of a gene or part of a gene in an antisense orientation may produce a desirable effect by preventing or inhibiting expression of an endogenous gene.
In order to produce a transgenic crop plant, a DNA construct that includes a heterologous gene sequence is introduced into a crop plant cell. The plant cell is then regenerated to produce a transgenic crop plant. The DNA construct includes a plant promoter that is operably linked to the heterologous gene sequence, often a promoter not normally associated with the heterologous gene. The promoter controls expression of the introduced DNA sequence to which the promoter is operably linked and thus affects the desired characteristic conferred by the DNA sequence. However, the exact level of expression of the new phenotype and its subsequent commercial utility in the crop plant is generally not known until the DNA construct has been successfully introduced into the target crop. Transgenic crop plants are usually difficult, time consuming and expensive to transform, with cotton and soybeans being leading examples. When a large number of DNA constructs need to be tested for efficacy in a particular crop plant, it requires many resources to produce the large number of plants necessary to adequately screen the DNA constructs for the most effective one or two. Alternatively, one could produce only a few plants with each DNA construct to study their properties and then follow that step with another round of transformation to obtain the large number of transformation events usually required to obtain one line with the desirable characteristics necessary for commercial success. This choice requires a longer time to reach the desired result, having two rounds of transformation in a crop plant. It would be advantageous to have a rapid method of testing these many DNA constructs and selecting from these a small number that will perform in a crop plant at a useful level. A large number of transgenic crop plants can then be produced from the selected few DNA constructs, thus increasing the probability of identifying the most useful lines.
There are many varieties of promoters and other regulatory elements that affect the transgene expression such that a gene or genes is transcribed efficiently at the right time during plant growth and development, in the optimal location the plant, and in the amount necessary to produce the desired effect. The level and rate of transcription of the transgene of interest is controlled by the various genetic elements of the transgene expression cassette. Promoters, introns, and leaders affect the expression level of the gene of interest, as well as the tissue localization, which is especially important for herbicide tolerance genes. It has been particularly difficult to predict the performance of DNA constructs for herbicide tolerance at a whole crop plant level. Low levels of tolerance in the reproductive parts of the plant can result in a plant that apparently survives the herbicide treatment but does not produce economically sufficient quantities of the product of interest such as oilseeds or fiber. This has resulted in the need to provide multiple combinations of genetic elements operably linked in DNA constructs, then testing these in crop plants for their ability to confer the desired herbicide tolerance phenotype. Generally, large numbers of transgenic crop plants have to be produced in order to select the individual lines with commercially acceptable herbicide tolerance, it is impractical to incur the time and expense of testing many DNA constructs in crop plants that result in no usable lines. In addition, increasingly stringent government regulatory requirements on the contents of DNA constructs used for plant transformation and the insertion of expression cassettes into the plant genome makes it necessary to select plant lines from a large number of candidate lines and include backup lines to comply with the requirements. In this regulatory environment, resources cannot be wasted on DNA constructs that do not provide the requisite attributes. Therefore, an unmet need in genetic engineering of plants for herbicide tolerance is a rapid method to screen large numbers of DNA constructs in a transgenic model plant system, one that reflects the performance of the DNA constructs in various target crop plants.
Additionally, the commercial development of genetically improved germplasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking approach. In this approach, multiple genes conferring different characteristics of interest can be introduced into a plant. It is important when introducing multiple genes into a plant that each gene has been selected from the many possible combinations of elements so that the desired phenotype can be effectively expressed in the target crop plant. It is apparent that there is a need for a model transgenic plant system that is indicative of the performance of a DNA construct in a target crop plant for stacking gene traits.
Arabidopsis thaliana has a long history of use as a model plant to test the expression pattern of individual promoters, usually by placing them 5′ to a reporter gene such as GUS, and the expression of transgenes. Also, many useful genes have been isolated from Arabidopsis and transferred to crop plant species. However, even though Arabidopsis genes have been isolated and transferred to other plants and DNA constructs having heterologous DNA sequences have been transformed into Arabidopsis, there has not been an effort to develop a system where Arabidopsis is used for the purpose of selecting from a number of possibly efficacious DNA constructs comprising a herbicide tolerance gene, a smaller number that would then be transformed into a crop plant of interest. The method of the present invention provides a process for selecting DNA constructs that have the greatest potential for producing herbicide tolerance in crop plants.